Methods and compositions relating to useful antigens of Moraxella catarrhalis

ABSTRACT

The present disclosure relates to selected antigenic proteins obtained from the outer membranes of Moraxella catarrhalis, that have been found by the inventors to have a variety of useful properties. These proteins, termed OMPs (&#34;Outer Membrane Proteins&#34;), are characterized as having molecular weights of 30, 80 and 100 kD, respectively. Studies set forth herein demonstrate that monoclonal antibodies directed against these proteins confer a protective effect against infection by Moraxella catarrhalis organisms in animal models, demonstrating the potential usefulness of such antibodies in conferring passive immunity as well as the potential usefulness of these OMPs, or variants thereof, in the preparation of vaccines. Also disclosed are DNA segments encoding these OMPs, methods for preparing the antigens, or variants, through the application of recombinant DNA techniques, as well as diagnostic methods and embodiments.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to various outer membraneproteins (OMPs) of Moraxella catarrhalis which have been found by theinventors to be useful targets in immunotherapy, such as in thepreparation of vaccines or protective antibodies for use in treatment ofMoraxella catarrhalis-related diseases. In particular aspects, thepresent invention concerns antigens identified by molecular weights of30, 80 and 100 kD, recombinant clones encoding these antigens, antigenfragments derived therefrom, equivalents thereof, as well as toantibodies reactive with these species. Further, the invention concernsmethods for the detection of Moraxella catarrhalis antigens andantibodies, as well as the use of specific antigens both in passive andactive immunity against Moraxella catarrhalis infections.

2. Description of the Related Art

It was previously thought that Moraxella catarrhalis (previously knownas Branhamella catarrhalis or Neisseria catarrhalis) was a harmlesssaprophyte of the upper respiratory tract. However, during the previousdecade, it has been determined that this organism is an important humanpathogen. In fact, recent studies have established this Gram-negativediplococcus as the cause of a number of human infections (Murphy, 1989).For example, Moraxella catarrhalis is a leading cause of otitis media,acute maxillary sinusitis as well as generalized infections of the lowerrespiratory tract (see, e.g., Murphy et al., 1989). Studies haveestablished that the incidence of otitis media and sinusitis attributedto Moraxella catarrhalis infections is increasing, with it being aboutthe third most common causative organism. In fact, reports haveidentified otitis media as the most common disease for which infants andchildren receive health care (Consensus, 1989).

The "Consensus" report referred to above concluded that prevention ofotitis media is an important health care goal due to both its occurrencein infants and children, as well as certain populations of all agegroups. In fact, the total financial burden of otitis media has beenestimated to be at least 2.5 billion annually, or approximately 3% ofthe health care budget. Vaccines were identified as the most desiredapproach to the prevention of this disease for a number of reasons. Forexample, it was estimated that if vaccines could reduce the incidence ofotitis media by 30%, this outcome could bring about an annual healthcare savings of at least $400 million. However, while some progress hasbeen made in the development of vaccines for 2 of the 3 common otitismedia pathogens, Streptococcus pneumoniae and Haemophilus influenzae,there is no indication that similar progress has been made with respectto Moraxella catarrhalis. This is particularly troublesome in thatMoraxella catarrhalis now accounts for approximately 17-20% of allotitis media infection (Murphy, 1989).

Previous attempts have been made to identify and characterize Moraxellacatarrhalis antigens that would serve as potentially important targetsof the human immune response to infection (Murphy, 1989; Goldblatt etal., 1990; Murphy et al., 1990). Generally speaking, the surface ofMoraxella catarrhalis is composed of outer membrane proteins (OMPs),lipooligo-saccharide (LOS) and fimbriae. As Murphy points out, Moraxellacatarrhalis appears to be somewhat distinct from other gram-negativebacteria in that attempts to isolate the outer membrane of this organismusing detergent fractionation of cell envelopes has generally proven tobe unsuccessful in that the procedures did not yield consistent results.Moreover, preparations were found to be contaminated with cytoplasmicmembranes which suggest an unusually characteristic of the Moraxellacatarrhalis cell envelope.

However, workers in the field have demonstrated the existence of 7 or 8major OMP species, and these appear to be fairly consistent fromMoraxella catarrhalis strain to strain, in spite of the great diversityof stains tested. For example, Campagnari et al. has identified the OMPsby letters A-H beginning with a band of molecular weight 98 Kd (OMP-A)and proceeding to the band with a molecular weight of about 21 Kd(OMP-H). (Campagnari et al., 1987).

The LOS of Moraxella catarrhalis has also been suggested as a possibletarget for vaccine development. LOS has been isolated from Moraxellacatarrhalis strains and subjected to SDS-PAGE and silver staining(Murphy, 1989). It was reported that all but one strain produced anidentical pattern of LOS staining. Thus, it appears that the LOS ofMoraxella catarrhalis is very highly antigenically conserved, thusraising the feasibility of using a portion of the LOS molecule as avaccine component.

Lastly, the Fimbriae have been suggested as a possible vaccinecandidate. Fimbriae apparently play a role in adherence and colonizationof mucosal services in some bacteria. Workers in the field havepostulated that if antigenically conserved epitopes are expressed onfimbriae and can be identified, then it is possible that antibodies tosuch epitopes might be useful therapeutically, or that such epitopes canserve as vaccine components.

Unfortunately, although various subcomponents of the Moraxellacatarrhalis cell have been suggested as places to begin a search forvaccine candidates, there has still been no such candidate identified.Certainly, no antigenic epitope or epitopes have been shown to induceprotective antibodies. Thus, it is clear that there is currently a needto identify which, if any, Moraxella catarrhalis component may serve asuseful antigens that can, for example, be employed in the preparation ofboth passive and active immunotherapeutic reagents such as vaccines.Additionally, once such an antigen or antigens is identified, there is aneed for providing methods and compositions which will allow thepreparation of these vaccines and quantities that will allow their useon a wide scale basis in therapeutic protocols.

SUMMARY OF THE INVENTION

Accordingly, in a general and overall sense, the present invention isconcerned with the identification and subsequent preparation of anMoraxella catarrhalis antigen species that would be of use both in theprevention and diagnosis of disease. In more particular terms, theinvention concerns the inventors' surprising discovery that particularMoraxella catarrhalis OMP antigens, including the 30, 80 and 100 Kd OMPantigens, have particular utility in vaccine development. It ispostulated by the inventors, therefore, these antigens can be useddirectly as a component of a vaccine, or can be employed for thepreparation of corresponding or equivalent antigen through sequenceanalysis.

It should be pointed out that of these OMP antigens, the inventorsbelieve that the 30 and 100 kD species will prove to be the most useful,in that their studies have shown that antibodies directed against thesetwo OMP species are broadly reactive with Moraxella catarrhalis subtypesand isolates. However, antibodies against the 80 kD species have not, asyet, been shown to react with all subspecies, and thus may not bepan-reactive. Thus, particularly preferred embodiments of the inventionconcern the 30 and 100 kD OMP antigens, DNA fragments encoding theseantigens and related species, antibodies recognizing these antigenspecies, and the like.

In certain embodiments, the present invention thus concerns an antigencomposition comprising a purified protein or peptide antigenincorporating an epitope that is immunologically cross-reactive with oneor more of the foregoing M. catarrhalis OMP antigens. While, generally,the purified protein or peptide antigen will comprise the OMP itself,the present disclosure provides techniques which may be employed forpreparing variants of these OMP antigens, peptides that incorporaterelated antigenic epitopes, as well as antigenic functional equivalentsof each of these. Furthermore, in that DNA segments encoding the variousOMP antigens are disclosed, the antigens may be provided essentiallyfree of antigenic epitopes from other M. catarrhalis antigens throughthe application of recombinant technology. That is, one may prepare theantigen by recombinant expression means using a host cell other than M.catarrhalis or related species, and thereby provide the antigen in anessentially pure antigenic state, with respect to other M. catarrhalisantigens. Such preparations will therefore be free, e.g., of LOS orfimbriae antigens.

In still further embodiments, through the use of standard DNA sequencingtechnology, DNA segments disclosed herein may be sequenced, and fromthis DNA sequence one may determine the underlying amino acid sequenceof the selected OMP protein, whether it be the 30, 80 or 100 kD species.Once this information is obtained, identification of suitable antigenicepitopes is a relatively straightforward matter through the use of, forexample, software programs for the prediction of such epitopes that areavailable to those of skill in the art. The amino acid sequence of these"epitopic core sequences" may then be readily incorporated into shorterpeptides, either through the application of peptide synthesis orrecombinant technology. Preferred peptides will generally be on theorder of 15 to 50 amino acids in length, and more preferably about 15 toabout 30 amino acids in length. It is proposed that shorter antigenicpeptides which incorporate epitopes of the selected OMP will provideadvantages in certain circumstances, for example, in the preparation ofvaccines or in immunologic detection assays. Exemplary advantagesinclude the ability to circumvent problems of contamination and purityoften associated with proteins prepared by recombinant production inthat peptides of this length may be prepared readily be synthetic meansusing peptide synthesizers.

In other embodiments, the present invention concerns processes forpreparing compositions which include purified protein or peptideantigens that incorporate epitopes that are immunologicallycross-reactive with the 30, 80 or 100 kD OMP. In a general sense, theseprocesses include first selecting cells that are capable of expressingsuch a protein or peptide antigen, culturing the cells under conditionseffective to allow expression of the antigen, and collecting the antigento thereby prepare the composition. Where one desires to prepare the OMPantigen itself, one will simply desire to culture M. catarrhalis cellsas a first step. In this case, the antigen will be provided, uponexpression, in the outer membrane fraction of the cell. The antigen isthen prepared by, first, preparation of membrane fraction followed bysolubilization and extraction of the antigen from the prepared membranesusing an ionic or nonionic detergent. Further purification may beachieved by a variety of methods including column fractionation,isoelectric focusing, and the like, or even immunoadsorption employingOMP-directed antibodies.

Of course, in light of the disclosure herein one may choose morepreferred embodiments to prepare the desired antigen that includeexpressing a recombinant DNA segment encoding the antigen in arecombinant host cell. Preferred recombinant host cells for expressionof antigens in accordance with the invention will typically be abacterial host cell in that the antigen is a bacterial antigen.Preferred bacterial host cells include E. coli, H. influenzae,Salmonella species, Mycobacterium species, or even Bacillus subtiliscells. Of course, where desired, one may also express the desiredantigen or antigens in eukaryotic cells.

As indicated above, in particular embodiments, the present inventionconcerns DNA segments which encode the desire protein or peptideantigen. Methods are disclosed herein for obtaining such segments in apurified state relative to their naturally occurring state. These DNAsegments will have a number of advantages and uses. For example,segments encoding the entire OMP gene may be introduced into recombinanthost cells and employed for expressing the entire protein antigen.Alternatively, through the application of genetic engineeringtechniques, subportions or derivatives of the selected OMP gene may beemployed to prepare shorter peptide sequences which neverthelessincorporate the desired antigenic epitopes. Furthermore, through theapplication of site-directed mutagenesis techniques, one may re-engineerDNA segments of the present invention to alter the coding sequence,e.g., to introduce improvements to the antigenicity of epitopic coresequences and thereby prepare antigenically functional equivalentpeptides. Of course, where desired, one may also prepare fusionpeptides, e.g., where the antigen coding regions are aligned within thesame expression unit with other desired antigen or proteins or peptideshaving desired functions, such as for immunodetection purposes (e.g.,enzyme label coding regions).

Depending on the host system employed, one may find particularadvantages where DNA segments of the present invention are incorporatedinto appropriate vector sequences which may, e.g., improve theefficiency of transfection of host cells. Where bacterial host cells areemployed, it is proposed that virtually any vector known in the art tobe appropriate for the selected host cell may be employed. Thus, in thecase of E. coli, one may find particular advantages through the use ofplasmid vectors such as pBR322, or bacteriophages such as λGEM-11. Otherparticular examples are disclosed hereinbelow.

In the preparation of recombinant clone banks from which appropriatelytransfected cells are selected, it will generally be the case thatexpression of the selected OMP gene sequences can be achieved in suchhost cells without the use of vectors having their own intrinsicpromoter sequences. This is because the genomic M. catarrhalis DNAfragments employed for clone bank preparation will include endogenouspromoters associated with the various coding sequences. However, theinventors propose that one may ultimately desire to re-engineer thepromoter region of the antigen-coding fragments of the present inventionto introduce heterologous promoter. This may allow one to overexpressthe OMP antigen in relation to its natural expression by M. catarrhaliscells.

It is contemplated that nucleic acid segments of the present inventionwill have numerous uses other than in connection with expression ofantigenic peptides or proteins. For example, nucleic acid segments of atleast 14 or so nucleotides in length that incorporate regions of the OMPgene sequence may be employed as selective hybridization probes for thedetection of M. catarrhalis sequences in selected samples or, e.g., toscreen clone banks to identify clones which comprise corresponding orrelated sequences. Furthermore, short segments may be employed asnucleic acid primers, such as in connection with PCR technology, for usein any of a number of applications, including, e.g., cloning andengineering exercises, or in PCR-based detection protocols.

In still further embodiments, the invention concerns the preparation ofantibodies capable of immunocomplexing with epitopes of the OMP antigen.Particular techniques for preparing antibodies in accordance with theinvention are disclosed hereinbelow. However, it is proposed by theinventors that any of the current techniques known in the art for thepreparation of antibodies in general may be employed, through theapplication of either monoclonal or polyclonal technology. As notedabove, a surprising aspect of the invention involves the inventors'discovery that monoclonal antibodies directed against the 30, 80 and 100kDa OMP antigens provide a protective effect against M. catarrhalischallenge in animal models. This surprising finding indicates not onlythat antibodies may be employed in the preparation of compositions foruse in connection with passive immunotherapy, but further, that epitopesof these OMP antigens may be employed in the preparation of vaccinecompositions. Accordingly, the present invention is directed both tovaccine compositions which include an antigen in accordance with thepresent invention, or antibodies against such an antigen, together witha pharmaceutically acceptable carrier, diluent, or adjuvant.

In still further embodiments, the present invention concernsimmunodetection methods and associated kits. It is proposed thatantigens of the present invention may be employed to detect antibodieshaving reactivity therewith, or, alternatively, antibodies prepared inaccordance with the present invention, may be employed to detectantigens. In general, these methods will include first obtaining asample suspected of containing such an antigen or antibody, contactingthe sample with an antibody or antigen in accordance with the presentinvention, as the case may be, under conditions effective to allow theantibody to form an immunocomplex with the antigen or antibody to bedetected, and detecting the presence of the antigen in the sample bydetecting the formation of an immunocomplex. In general, the detectionof immunocomplex formation is quite well known in the art and may beachieved through the application of numerous approaches. For example,the present invention contemplates the application of ELISA, RIA,immunoblot, dotblot, indirect immunofluorescence techniques and thelike. Generally, immunocomplex formation will be detected through theuse of a label, such as a radiolabel or an enzyme tag (such as alkalinephosphatase, horseradish peroxidase, or the like). Of course, one mayfind additional advantages through the use of a secondary binding ligandsuch as a second antibody or a biotin/avidin ligand binding arrangement,as is known in the art.

For diagnostic purposes, it is proposed that virtually any samplesuspected of comprising either the antigen or antibody sought to bedetected, as the case may be, may be employed. Exemplary samples includeclinical samples obtained from a patient such as blood or serum samples,ear swabs, sputum samples, middle ear fluid or even perhaps urinesamples may be employed. Furthermore, it is contemplated that suchembodiments may have application to non-clinical samples, such as in thetitering of antigen or antibody samples, in the selection of hybridomas,and the like.

In related embodiments, the present invention contemplates thepreparation of kits that may be employed to detect the presence ofantigens and/or antibodies in a sample. Generally speaking, kits inaccordance with the present invention will include a suitable OMPantigen (i.e., either the 30, 80 or 100 kD species, or proteincontaining epitopes corresponding to one or more of these species), orantibody directed against such an antigen, together with animmunodetection reagent and a means for containing the antibody orantigen and reagent. The immunodetection reagent will typically comprisea label associated with the antibody or antigen, or associated with asecondary binding ligand. Exemplary ligands might include a secondaryantibody directed against the first antibody or antigen or a biotin oravidin (or streptavidin) ligand having an associated label. Of course,as noted above, a number of exemplary labels are known in the art andall such labels may be employed in connection with the presentinvention.

The container means will generally include a vial into which theantibody, antigen or detection reagent may be placed, and preferablysuitably aliquoted. The kits of the present invention will alsotypically include a means for containing the antibody, antigen, andreagent containers in close confinement for commercial sale. Suchcontainers may include injection or blow-molded plastic containers intowhich the desired vials are retained.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Western blot analysis of M. catarrhalis proteins using as aprobe monoclonal antibody 10F3, which recognizes the 80 kD OMP. Lane Ais a Rainbow protein molecular weight marker (M.W. 14.3 to 200 kD,Amersham); Lane B is a negative control comprising a whole cell lysateof 4B1/pBR322/RR1 (4B1 is an M. catarrhalis gene encoding an unrelatedprotein recognized by monoclonal antibody 4B1); Lanes C and D are wholecell lysates of 10F3/pBR322/RR1; and Lane E is a blank control.

FIG. 2. Western blot analysis of M. catarrhalis proteins using as aprobe monoclonal antibody 8B6, which recognizes the 30 kD OMP. Lane A isa Rainbow protein molecular weight marker (M.W. 14.3 to 200 kD,Amersham); Lane B is a prestained SDS-PAGE-standard, low molecularweight (M.W. 16 to 110 kD, Bio-Rad); Lane C contains proteins from aphage lysate of recombinant E. coli that express the 30 kD OMP(LE392/8B6); Lane D is a blank control; Lane E is a negative control(phage lysate from recombinant E. coli expressing the 100 kD OMP,LE392/17C7); and Lane F is a positive control (M. catarrhalis 035E outermembrane vesicles).

FIG. 3. Western blot analysis of M. catarrhalis proteins using as aprobe monoclonal antibody 17C7, which recognizes the 100 kD OMP. Lane Ais a Rainbow protein molecular weight marker (M.W. 14.3 to 200 kD,Amersham); Lane B is a prestained SDS-PAGE-standard, low molecularweight (M.W. 16 to 110 kD, Bio-Rad); Lanes C, D and E contain proteinsfrom a phage lysate of recombinant E. coli that express the 100 kD OMP(LE392/17C7); Lane F is a blank control; Lane H is a negative control(phage lysate from recombinant E. coli expressing the 30 kD OMP, E.coli/8B6 phage lysate); and Lane G is a positive control (M. catarrhalis035E outer membrane vesicles).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to the inventors' identification ofparticular outer membrane proteins (OMPs) of Moraxella catarrhalis thatare found to have particularly useful properties, e.g., in thepreparation of both diagnostic and therapeutic reagents. These proteinsappear to be cell surface-exposed in their natural state, and exhibitmolecular weights of about 30, 80 and 100 kilodaltons, respectively,upon SDS-PAGE. Particular embodiments relate to the recombinant cloningof sequences encoding these proteins, antigenic subfragments, variants,and the like. The present invention also relates to monoclonalantibodies to these M. catarrhalis OMPs that are shown to reduce thenumber of infecting M. catarrhalis bacteria present in localized lunginfections, as demonstrated in pulmonary clearance studies using amurine model system.

Recombinant clones, expressing one or more of the selected OMPs, andthat may be used to prepare purified OMP antigens as well as mutant orvariant protein species in significant quantities, are included withinthe scope of the disclosure. The selected OMP antigen, and variantsthereof, are anticipated to have significant utility in diagnosing andtreating M. catarrhalis infections. For example, it is proposed thatthese OMP antigens, or peptide variants, may be used in immunoassays todetect M. catarrhalis or as a vaccine to treat M. catarrhalisinfections.

As those skilled in the art will appreciate, the nucleic acid sequenceswhich encode for the selected OMP antigen, or their variants, may beuseful in hybridization or polymerase chain reaction (PCR) methodologyto detect M. catarrhalis. Accordingly, included in the present inventiondisclosure is information which may be used to prepare a wide variety ofDNA fragments having a number of potential utilities, such as thepreparation of relatively short immunogenic/antigenic peptidylsubfragments of the antigen, the use of DNA or RNA sequences in PCR andhybridization studies as probes for in vitro detection, as well as otheruseful medical and biomedical applications related to the research,diagnosis and treatment of M. catarrhalis infections.

The OMP antigens of the present invention are referred to, respectively,as the 30, 80 and 100 kD OMPs. These proteins have been identified bythe inventors by reference to monoclonal antibodies that were selectedfrom a battery of monoclonal antibodies against M. catarrhalis outermembrane vesicles. These antibodies were employed as Western blot probesto identify corresponding antigens from SDS-PAGE runs of M. catarrhalis035E outer membrane vesicle preparations. The monoclonal antibodyrecognizing the 30 kD OMP is termed 8B6, the antibody recognizing the 80kD OMP is termed 10F3, and that recognizing the 100 kD antigen has beendesignated 17C7 (see FIGS. 1 through 3). Importantly, each of theforegoing hybridomas have been shown to be protective against M.catarrhalis infection in animal models.

The present invention envisions various means for both producing andisolating the OMP antigen proteins of the present invention, rangingfrom isolation of purified or partially purified protein from naturalsources (e.g., from M. catarrhalis bacterial cells), or from recombinantDNA sources (e.g., E. coli or microbial cells). In the latter case, theOMP antigens of the invention, or antigenic peptides derived therefrom,may be provided in essentially antigenically pure states in that theywill be free of other M. catarrhalis epitopes unrelated to the selectedOMP species.

It is proposed that isolation of the OMP antigen from either natural orrecombinant sources in accordance with the invention may be achievedisolating cell envelopes or outer membranes and then using adetergent-based purification scheme. In the case of recombinant cells,the desired antigen may be present in inclusion bodies.

Since monoclonal antibodies to the 30, 80 and 100 kD OMP antigens aredisclosed by the present invention, the use of immunoabsorbanttechniques are anticipated to be useful in purifying the OMP antigen, orits immunologically cross reactive variants. It is proposed that usefulantibodies for this purpose may be prepared generally by the techniquesdisclosed hereinbelow, or as in generally known in the art for thepreparation of monoclonals (see, e.g., U.S. Pat. Nos. 4,514,498 and4,740,467), and those reactive with the desired OMP protein or peptidesselected. Moreover, it is believed that the foregoing general isolationscheme will work equally well for isolation of OMP variants or ofantigenic/immunogenic subfragments of the protein, requiring only thegeneration and use of antibodies having affinity for the desiredpeptidyl region.

Additionally, by application of techniques such as DNA mutagenesis, thepresent invention allows the ready preparation of so-called "secondgeneration" molecules having modified or simplified protein structures.Second generation proteins will typically share one or more propertiesin common with the full-length antigen, such as a particularantigenic/immunogenic epitopic core sequence. Epitopic sequences can beprovided on relatively short molecules prepared from knowledge of thepeptide, or underlying DNA sequence information. Such variant moleculesmay not only be derived from selected immunogenic/antigenic regions ofthe protein structure, but may additionally, or alternatively, includeone or more functionally equivalent amino acids selected on the basis ofsimilarities or even differences with respect to the natural sequence.

Epitopic Core Sequences of the OMP Antigens

As noted above, it is proposed that particular advantages may berealized through the preparation of synthetic peptides which includeepitopic/immunogenic core sequences. These epitopic core sequences areidentified herein in particular aspects as hydrophilic regions of theOMP antigen. It is proposed that these regions represent those which aremost likely to promote T-cell or B-cell stimulation, and, hence, elicitspecific antibody production. An epitopic core sequence, as used herein,is a relatively short stretch of amino acids that is "complementary" to,and therefore will bind, antigen binding sites on OMP-directedantibodies. Additionally or alternatively, an epitopic core sequence isone that will elicit antibodies that are cross-reactive with OMPdirected antibodies. It will be understood that in the context of thepresent disclosure, the term "complementary" refers to amino acids orpeptides that exhibit an attractive force towards each other. Thus,certain epitope core sequences of the present invention may beoperationally defined in terms of their ability to compete with orperhaps displace the binding of the desired OMP antigen with thecorresponding OMP-directed antisera.

In general, the size of the polypeptide antigen is not believed to beparticularly crucial, so long as it is at least large enough to carrythe identified core sequence or sequences. The smallest useful coresequence anticipated by the present disclosure would be on the order ofabout 15 amino acids in length. Thus, this size will generallycorrespond to the smallest peptide antigens prepared in accordance withthe invention. However, the size of the antigen may be larger wheredesired, so long as it contains a basic epitopic core sequence.

Accordingly, through the use of computerized peptide sequence analysisprogram (DNAStar Software, DNAStar, Inc., Madison, Wis.), the inventorproposes to identify particular hydrophilic peptidyl regions of the 30,80 or 100 kD OMP antigen which are believed to constitute epitopic coresequences comprising particular epitopes of the protein.

Syntheses of epitopic sequences, or peptides which include an antigenicepitope within their sequence, are readily achieved using conventionalsynthetic techniques such as the solid phase method (e.g., through theuse of commercially available peptide synthesizer such as an AppliedBiosystems Model 430A Peptide Synthesizer). Peptide antigens synthesizedin this manner may then be aliquoted in predetermined amounts and storedin conventional manners, such as in aqueous solutions or, even morepreferably, in a powder or lyophilized state pending use.

In general, due to the relative stability of peptides, they may bereadily stored in aqueous solutions for fairly long periods of time ifdesired, e.g., up to six months or more, in virtually any aqueoussolution without appreciable degradation or loss of antigenic activity.However, where extended aqueous storage is contemplated it willgenerally be desirable to include agents including buffers such as Trisor phosphate buffers to maintain a pH of 7.0 to 7.5. Moreover, it may bedesirable to include agents which will inhibit microbial growth, such assodium azide or Merthiolate. For extended storage in an aqueous state itwill be desirable to store the solutions at 4° C., or more preferably,frozen. Of course, where the peptide(s) are stored in a lyophilized orpowdered state, they may be stored virtually indefinitely, e.g., inmetered aliquots that may be rehydrated with a predetermined amount ofwater (preferably distilled) or buffer prior to use.

Antigenically Functional Equivalent Amino Acids

As noted above, it is believed that numerous modifications and changesmay be made in the structure of the desired OMP antigen, orantigenic/immunogenic subportions thereof, and still obtain a moleculehaving like or otherwise desirable characteristics.

It is, for example, known that certain amino acids may be substitutedfor other amino acids in a protein structure in order to modify orimprove its antigenic or immunogenic activity (see, e.g., Kyte et al, orHopp, U.S. Pat. No. 4,554,101, incorporated herein by reference). Forexample, through the substitution of alternative amino acids, smallconformational changes may be conferred upon an antigenic peptide whichresult in increase affinity between the antigen and the antibody bindingregions. Alternatively, amino acid substitutions in certain OMPantigenic peptides may be utilized to provide residues which may then belinked to other molecules to provide peptide-molecule conjugates whichretain enough antigenicity of the starting peptide to be useful forother purposes. For example, a selected OMP peptide bound to a solidsupport might be constructed which would have particular advantages indiagnostic embodiments.

The importance of the hydropathic index of amino acids in conferringinteractive biologic function on a protein has been discussed generallyby Kyte et al. (1982), wherein it is found that certain amino acids maybe substituted for other amino acids having a similar hydropathic indexor core and still retain a similar biological activity. As displayed inthe table below, amino acids are assigned a hydropathic index on thebasis of their hydrophobicity and charge characteristics. It is believedthat the relative hydropathic character of the amino acid determines thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with substrate molecules. Preferredsubstitutions for monitoring binding capability will generally involveamino acids having index scores within 2 units of one another, and morepreferably within 1 unit.

                  TABLE I                                                         ______________________________________                                        Amino Acid     Hydropathic Index                                              ______________________________________                                        Isoleucine     4.5                                                            Valine         4.2                                                            Leucine        3.8                                                            Phenylalanine  2.8                                                            Cysteine/cystine                                                                             2.5                                                            Methionine     1.9                                                            Alanine        1.8                                                            Glycine        -0.4                                                           Threonine      -0.7                                                           Tryptophan     -0.9                                                           Serine         -0.8                                                           Tyrosine       -1.3                                                           Proline        -1.6                                                           Histidine      -3.2                                                           Glutamic Acid  -3.5                                                           Glutamine      -3.5                                                           Aspartic Acid  -3.5                                                           Asparagine     -3.5                                                           Lysine         -3.9                                                           Arginine       -4.5                                                           ______________________________________                                    

Thus, for example, isoleucine, which has a hydropathic index of +4.5,will preferably be exchanged with an amino acid such as valine (+4.2) orleucine (+3.8). Alternatively, at the other end of the scale, lysine(-3.9) will preferably be substituted for arginine (-4.5), and so on.

Accordingly, these amino acid substitutions are generally based on therelative similarity of R-group substituents, for example, in terms ofsize, electrophilic character, charge, and the like. In general,preferred substitutions which take various of the foregoingcharacteristics into consideration include the following:

                  TABLE II                                                        ______________________________________                                        Original Residue                                                                            Exemplary Substitutions                                         ______________________________________                                        Ala           gly; ser                                                        Arg           lys                                                             Asn           gln; his                                                        Asp           glu                                                             Cys           ser                                                             Gln           asn                                                             Glu           asp                                                             Gly           ala                                                             His           asn; gln                                                        Ile           leu; val                                                        Leu           ile; val                                                        Lys           arg; gln; glu                                                   Met           leu; ala                                                        Ser           thr                                                             Thr           ser                                                             Trp           tyr                                                             Tyr           trp; phe                                                        Val           ile; leu                                                        ______________________________________                                    

Preparation of Monoclonal Antibodies to M. catarrhalis OMPs

Monoclonal antibodies specific for the Moraxella catarrhalis OMPs of thepresent invention may be prepared using conventional immunizationtechniques. Initially, a composition containing antigenic epitopes ofthe OMP, such as an outer membrane vesicle preparation, can be used toimmunize an experimental animal, such as a mouse, from which apopulation of spleen or lymph cells are subsequently obtained. Thespleen or lymph cells can then be fused with cell lines, such as humanor mouse myeloma strains, to produce antibody-secreting hybridomas.These hybridomas may be isolated to obtain individual clones which canthen be screened for production of antibody to the desired OMP.

In particular aspects, the present invention utilizes outer membranefragments from M. catarrhalis to induce an immune response inexperimental animals. Following immunization, spleen cells are removedand fused, using a standard fusion protocol (see, e.g., The Cold SpringHarbor Manual for Hybridoma Development, incorporated herein byreference) with plasmacytoma cells to produce hybridomas secretingmonoclonal antibodies against outer membrane proteins. Hybridomas whichproduce monoclonal antibodies to the selected OMP are identified usingstandard techniques, such as ELISA and Western blot methods.

Hybridoma clones can then be cultured in liquid media and the culturesupernatants purified to provide the OMP-specific monoclonal antibodies.

Use of Monoclonal Antibodies to OMP Antigens

In general, monoclonal antibodies to the desired OMP antigen of M.catarrhalis can be used in both the diagnosis and treatment of M.catarrhalis infections.

It is proposed that the monoclonal antibodies of the present inventionwill find useful application in standard immunochemical procedures, suchas ELISA and Western blot methods, as well as other procedure which mayutilize antibody specific to OMP epitopes. These OMP-specific monoclonalantibodies are anticipated to be useful in various ways for thetreatment of M. catarrhalis infections through, for example, theirapplication in passive immunization procedures.

Additionally, it is proposed that monoclonal antibodies specific to theparticular OMP may be utilized in other useful applications. Forexample, their use in immunoabsorbent protocols may be useful inpurifying native or recombinant OMP species or variants thereof.

Studies have shown that antibody preparations against the OMP antigensof the invention have a significant protective effect against M.catarrhalis infection. The present inventors have shown that passiveimmunization with monoclonal antibodies specific for these OMPssignificantly reduce the numbers of M. catarrhalis organisms following abolus injection of bacteria. This demonstrates that these OMP antigensmay be employed in making gammaglobulin preparations for use in passiveimmunization against disorders associated with M. catarrhalisinfections, or used directly as vaccine components.

To obtain suitable gammaglobulin preparations, one may desire to preparemonoclonal antibodies, preferably human or humanized hybridomas.Alternatively, it is proposed that one may desire to use globulinfractions from hyperimmunized individuals.

Recombinant Cloning Genes Encoding M. catarrhalis OMPs

The present invention also involves isolating M. catarrhalis OMP genes,or sequence variants, incorporating DNA segments encoding the 30, 80 or100 kD OMP gene into a suitable vector, and transforming a suitablehost, such that recombinant proteins, or variants thereof, areexpressed. It will be appreciated by those of skill in the art that inlight of the present disclosure the invention is also applicable to theisolation and use of the OMP gene sequences from any suitable sourcethat includes appropriate coding sequences, such as any M. catarrhalissubspecies or isolate that expresses the desired OMP. Such sources maybe readily identified by immunological screening with monoclonalantibodies to the selected OMP.

The preferred application of the present invention to the isolation anduse of OMP-encoding DNA involves generally the steps of (1) isolation ofMoraxella genomic DNA; (2) partial restriction enzyme digestion of thegenomic DNA with an enzyme such as PstI, (the selected restrictionenzyme is not crucial) to provide DNA having an average length of, e.g.,6 to 23 kb; (3) ligation of the partially digested DNA into a selectedsite within a selected vector, such as pBR322 (again, other plasmid orphage vectors may be used at this step, as desired); (4) transformation,transfection or electroporation of suitable host cells, e.g., E. colicells, with the recombinant vector; and (5) selection of coloniesexpressing the desired OMP through the application of specificallydesigned screening protocols. Following identification of a clone whichcontains the OMP gene, one may desire to reengineer the gene into apreferred host/vector/promoter system for enhanced production of theouter membrane protein, or sequence variants thereof.

Through application of the foregoing general steps, the inventors havesucceeded in identifying and selecting a number of clones which containM. catarrhalis OMP genes in a manner which allows it to produce thecorresponding outer membrane protein.

In a preferred application of these techniques, genomic DNA fromMoraxella catarrhalis strain 035E was isolated from bacteria through theuse of SDS, ribonuclease and proteinase K treatment,phenol/chloroform/isoamyl alchohol extraction and ethanol precipitation.Conditions were determined for achieving an appropriate partialrestriction enzyme digestion, such as would provide fragments on theorder of 6-23 kb in length, using a restriction enzyme, such as PstI.After size fractionation, the partially digested Moraxella DNA fragmentsof the selected size range were ligated with fully digested vector, suchas pBR322, which was fully digested with PstI to generate compatiblesites for ligation with the genomic DNA fragments.

Following the ligation, the recombinant vectors are then used totransform a suitable host, such as E. coli RR1, to produce a recombinantlibrary having members that express M. catarrhalis protein speciesencoded by the DNA fragment inserts. The recombinant microbial clonesare cultivated, preferably on the surface of a nutrient agar, to formvisible colonies. Those colonies expressing surface-exposed M.catarrhalis outer membrane proteins are then identified using monoclonalantibodies to M. catarrhalis OMPs in a colony blot radioimmunoassay.Recombinant E. coli clones expressing proteins having epitopes reactivewith anti-OMP antibodies may then be cultured in desired quantities.

Host Cell Cultures and Vectors

In general, of course, prokaryotes are preferred for the initial cloningof DNA sequences and constructing the vectors useful in the invention.For example, E. coli strain RR1 is particularly useful. Other microbialstrains which may be used include E. coli strains such as E. coli LE392,E. coli B, and E. coli X 1776 (ATCC No. 31537). These examples are, ofcourse, intended to be illustrative rather than limiting.

Prokaryotes are also preferred for expression. The aforementionedstrains, as well as E. coli W3110 (F-, lambda-, prototrophic, ATCC No.273325), bacilli such as Bacillus subtilis, or other enterobacteriaceaesuch as Salmonella typhimurium or Serratia marcescens, and variousPseudomonas species may be used.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies (Bolivar et al., 1977). pBR322 contains genes for ampicillin andtetracycline resistance and thus provides easy means for identifyingtransformed cells. The pBR plasmid, or other microbial plasmid or phagemust also contain, or be modified to contain, promoters which can beused by the microbial organism for expression of its own proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used as atransforming vector in connection with these hosts. For example, thephage lambda GEM™-11 may be utilized in making recombinant phage vectorwhich can be used to transform host cells, such as E. coli LE392.

Those promoters most commonly used in recombinant DNA constructioninclude the B-lactamase (penicillinase) and lactose promoter systems(Chang et al., 1978; Itakura et al., 1977; Goeddel et al., 1979) and atryptophan (trp) promoter system (Goeddel et al., 1980; EPO Appl. Publ.No. 0036776). While these are the most commonly used, other microbialpromoters have been discovered and utilized, and details concerningtheir nucleotide sequences have been published, enabling a skilledworker to ligate them functionally with plasmid vectors (EPO Appl. Publ.No. 0036776).

In addition to prokaryotes, eukaryotic microbes, such as yeast culturesmay also be used. Saccharomyces cerevisiae, or common baker's yeast isthe most commonly used among eukaryotic microorganisms, although anumber of other strains are commonly available. For expression inSaccharomyces, the plasmid YRp7, for example, is commonly used(Stinchcomb et al., 1979; Kingsman et al., 1979; Tschemper et al.,1980). This plasmid already contains the trpl gene which provides aselection marker for a mutant strain of yeast lacking the ability togrow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, 1977).The presence of the trpl lesion as a characteristic of the yeast hostcell genome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzeman et al., 1980) or other glycolyticenzymes (Hess et al., 1968; Holland et al., 1978), such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also ligated into the expression vector 3' of the sequencedesired to be expressed to provide polyadenylation of the mRNA andtermination. Other promoters, which have the additional advantage oftranscription controlled by growth conditions are the promoter regionfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and theaforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Any plasmid vectorcontaining a yeast-compatible promoter, origin of replication andtermination sequences is suitable.

In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (tissue culture) has become aroutine procedure in recent years (Tissue Culture, 1973). Examples ofsuch useful host cell lines are VERO and HeLa cells, Chinese hamsterovary (CHO) cell lines, and W138, BHK, COS-7, 293 and MDCK cell lines.Expression vectors for such cells ordinarily include (if necessary) anorigin of replication, a promoter located in front of the gene to beexpressed, along with any necessary ribosome binding sites, RNA splicesites, polyadenylation site, and transcriptional terminator sequences.

For use in mammalian cells, the control functions on the expressionvectors are often provided by viral material. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, and most frequentlySimian Virus 40 (SV40). The early and late promoters of SV40 virus areparticularly useful because both are obtained easily from the virus as afragment which also contains the SV40 viral origin of replication (Fierset al., 1978). Smaller or larger SV40 fragments may also be used,provided there is included the approximately 250 bp sequence extendingfrom the Hind III site toward the Bg1 I site located in the viral originof replication. Further, it is also possible, and often desirable, toutilize promoter or control sequences normally associated with thedesired gene sequence, provided such control sequences are compatiblewith the host cell systems.

As origin of replication may be provided either by construction of thevector to include an exogenous origin, such as may be derived from SV40or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or may beprovided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter is oftensufficient.

Sequencing of OMP Genes

After cloning the gene encoding the selected OMP, one will desire toperform restriction mapping and DNA sequence analysis, e.g., by thedideoxy method of Sanger et al. (1977). Both the DNA and the deducedamino acid sequence can then be compared with known sequences todetermine homologies with known proteins. The amino acid sequence of theprotein will reveal the nature of the various domains, e.g.,cytoplasmic, membrane-spanning, and substrate binding domains, and giveimportant information in terms of approaches to improving the structureof the enzyme through genetic engineering techniques.

Through the use of computerized peptide sequence analysis program(DNAStar Software, DNAStar, Inc., Madison, Wis.), particular hydrophilicpeptidyl regions of the OMP antigen may be identified which are likelyto constitute epitopic core sequences, comprising particular epitopes ofthe protein, as well as biologically functional equivalents of theforegoing peptides, as explained in more detail below.

Preparation of OMP Variants

Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent proteins orpeptides, derived from the OMP antigen sequence, through specificmutagenesis of the underlying DNA. The technique further provides aready ability to prepare and test sequence variants, for example,incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known inthe art as exemplified by publications (Adelman et al., 1983). As willbe appreciated, the technique typically employs a phage vector whichexists in both a single stranded and double stranded form. Typicalvectors useful in site-directed mutagenesis include vectors such as theM13 phage (Messing et al., 1981). These phage are readily commerciallyavailable and their use is generally well known to those skilled in theart.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector which includeswithin its sequence a DNA sequence which encodes the OMP antigen. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically, for example by the method of Crea et al.(1978). This primer is then annealed with the single-stranded vector,and subjected to DNA polymerizing enzymes such as E. coli polymerase IKlenow fragment, in order to complete the synthesis of themutation-bearing strand. Thus, a heteroduplex is formed wherein onestrand encodes the original non-mutated sequence and the second strandbears the desired mutation. This heteroduplex vector is then used totransform appropriate cells, such as E. coli cells, and clones areselected which include recombinant vectors bearing the mutated sequencearrangement.

The preparation of sequence variants of the selected OMP gene usingsite-directed mutagenesis is provided as a means of producingpotentially useful OMP species and is not meant to be limiting as thereare other ways in which sequence variants of the OMP may be obtained.For example, recombinant vectors encoding the desired OMP gene may betreated with mutagenic agents to obtain sequence variants (see, e.g., amethod described by Eichenlaub, 1979) for the mutagenesis of plasmid DNAusing hydroxylamine.

Use of Nucleic Acid Sequences

As mentioned, in certain aspects, the DNA sequence information providedby the present disclosure allows for the preparation of relatively shortDNA (or RNA) sequences having the ability to specifically hybridize togene sequences of the selected OMP antigen gene. In these aspects,nucleic acid probes of an appropriate length are prepared based on aconsideration of the natural sequence or derived from flanking regionsof the OMP gene, such as regions downstream of the gene as found inplasmid pBR322. The ability of such nucleic acid probes to specificallyhybridize to OMP gene sequences lend them particular utility in avariety of embodiments. Most importantly, the probes can be used in avariety of diagnostic assays for detecting the presence of pathogenicorganisms in a given sample. However, other uses are envisioned,including the use of the sequence information for the preparation ofmutant species primers, or primers for use in preparing other geneticconstructions.

To provide certain of the advantages in accordance with the invention,the preferred nucleic acid sequence employed for hybridization studiesor assays includes sequences that are complementary to at least a 10 to20, or so, nucleotide stretch of the sequence. A size of at least 10nucleotides in length helps to ensure that the fragment will be ofsufficient length to form a duplex molecule that is both stable andselective. Molecules having complementary sequences over stretchesgreater than 10 bases in length are generally preferred, though, inorder to increase stability and selectivity of the hybrid, and therebyimprove the quality and degree of specific hybrid molecules obtained.Thus, one will generally prefer to design nucleic acid molecules havingOMP gene-complementary stretches of 15 to 20 nucleotides, or even longerwhere desired. Such fragments may be readily prepared by, for example,directly synthesizing the fragment by chemical means, by application ofnucleic acid reproduction technology, such as the PCR technology of U.S.Pat. No. 4,603,102, or by introducing selected sequences intorecombinant vectors for recombinant production.

In that the OMP antigens of the present invention are believed to beindicative of pathogenic Moraxella species, the present invention willfind particular utility as the basis for diagnostic hybridization assaysfor detecting OMP-specific RNA or DNA in clinical samples. Exemplaryclinical samples that can be used in the diagnosis of infections arethus any samples which could possibly include Moraxella nucleic acid,including middle ear fluid, sputum, bronchoalveolar fluid, amnioticfluid or the like. A variety of hybridization techniques and systems areknown which can be used in connection with the hybridization aspects ofthe invention, including diagnostic assays such as those described inFalkow et al., U.S. Pat. No. 4,358,535.

Accordingly, the nucleotide sequences of the invention are important fortheir ability to selectively form duplex molecules with complementarystretches of the corresponding OMP genes. Depending on the applicationenvisioned, one will desire to employ varying conditions ofhybridization to achieve varying degrees of selectivity of the probetoward the target sequence. For applications requiring a high degree ofselectivity, one will typically desire to employ relatively stringentconditions to form the hybrids, for example, one will select relativelylow salt and/or high temperature conditions, such as provided by0.02M-0.15M NaCl at temperatures of 50° C. to 70° C. These conditionsare particularly selective, and tolerate little, if any, mismatchbetween the probe and the template or target strand.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template, less stringent hybridization conditions are calledfor in order to allow formation of the heteroduplex. In thesecircumstances, one would desire to employ conditions such as 0.15M-0.9Msalt, at temperatures ranging from 20° C. to 55° C. In any case, it isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide, which serves todestabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

In certain embodiments, one may desire to employ nucleic acid probes toisolate variants from clone banks containing mutated clones. Inparticular embodiments, mutant clone colonies growing on solid mediawhich contain variants of the OMP sequence could be identified onduplicate filters using hybridization conditions and methods, such asthose used in colony blot assays, to only obtain hybridization betweenprobes containing sequence variants and nucleic acid sequence variantscontained in specific colonies. In this manner, small hybridizationprobes containing short variant sequences of the OMP gene may beutilized to identify those clones growing on solid media which containsequence variants of the entire OMP gene. These clones can then be grownto obtain desired quantities of the variant OMP nucleic acid sequencesor the corresponding OMP antigen.

In clinical diagnostic embodiments, nucleic acid sequences of thepresent invention are used in combination with an appropriate means,such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including radioactive,enzymatic or other ligands, such as avidin/biotin, which are capable ofgiving a detectable signal. In preferred diagnostic embodiments, onewill likely desire to employ an enzyme tag such as urease, alkalinephosphatase or peroxidase, instead of radioactive or other environmentalundesirable reagents. In the case of enzyme tags, colorimetric indicatorsubstrates are known which can be employed to provide a means visible tothe human eye or spectrophoto-metrically, to identify specifichybridization with pathogen nucleic acid-containing samples.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridizations aswell as in embodiments employing a solid phase. In embodiments involvinga solid phase, the test DNA (or RNA) from suspected clinical samples,such as exudates, body fluids (e.g., amniotic fluid, middle eareffusion, bronchoalveolar lavage fluid) or even tissues, is adsorbed orotherwise affixed to a selected matrix or surface. This fixed,single-stranded nucleic acid is then subjected to specific hybridizationwith selected probes under desired conditions. The selected conditionswill depend on the particular circumstances based on the particularcriteria required (depending, for example, on the G+C contents, type oftarget nucleic acid, source of nucleic acid, size of hybridizationprobe, etc.). Following washing of the hybridized surface so as toremove nonspecifically bound probe molecules, specific hybridization isdetected, or even quantified, by means of the label.

In other embodiments, it is proposed that OMP sequences or variantsthereof may be used to provide highly specific and sensitive detectionof M. catarrhalis when used as reagents in polymerase chain reaction(PCR) assays. In general, by applying the PCR technology as set out,e.g., in U.S. Pat. No. 4,60,102, one may utilize various portions of theOMP sequence as oligonucleotide probes for the PCR amplification of adefined portion of OMP nucleic acid in a sample. The amplified portionof the OMP sequence may then be detected by hybridization with ahybridization probe containing a complementary sequence. In this manner,extremely small concentrations of M. catarrhalis nucleic acid maydetected in a sample utilizing OMP sequences.

In other embodiments, OMP sequences may be utilized in PCR formats forthe in vitro preparation of desired quantities of selected portions ofthe OMP gene. By amplifying selected gene portions of a selected OMPgene and then incorporating those portions into vectors, one can alsoprepare recombinant clones which express OMP variants, includingsubfragments of the OMP antigen. In this manner, peptides carryingantigen epitopes of the outer membrane protein may be prepared andutilized for various purposes.

Immunoassays

As noted, it is proposed that OMP peptides of the invention will findutility as immunogens, e.g., in connection with vaccine development, oras antigens in immunoassays for the detection of anti-OMPantigen-reactive antibodies. Turning first to immunoassays, in theirmost simple and direct sense, preferred immunoassays of the inventioninclude the various types of enzyme linked immunosorbent assays (ELISAs)known to the art. However, it will be readily appreciated that theutility of OMP peptides is not limited to such assays, and that otheruseful embodiments include RIAs and other non-enzyme linked antibodybinding assays or procedures.

In the preferred ELISA assay, peptides incorporating OMP antigensequences are immobilized onto a selected surface, preferably a surfaceexhibiting a protein affinity such as the wells of a polystyrenemicrotiter plate. After washing to remove incompletely adsorbedmaterial, one will desire to bind or coat a nonspecific protein such asbovine serum albumin (BSA) or casein onto the well that is known to beantigenically neutral with regard to the test antisera. This allows forblocking of nonspecific adsorption sites on the immobilizing surface andthus reduces the background caused by nonspecific binding of antiseraonto the surface.

After binding of antigenic material to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with theantisera or clinical or biological extract to be tested in a mannerconducive to immune complex (antigen/antibody) formation. Suchconditions preferably include diluting the antisera with diluents suchas BSA, bovine gamma globulin (BGG) and phosphate buffered saline(PBS)/Tween. These added agents also tend to assist in the reduction ofnonspecific background. The layered antisera is then allowed to incubatefor from 2 to 4 hours, at temperatures preferably on the order of 25° to27° C. Following incubation, the antisera-contacted surface is washed soas to remove non-immunocomplexed material. A preferred washing procedureincludes washing with a solution such as PBS/Tween, or borate buffer.

Following formation of specific immunocomplexes between the test sampleand the bound antigen, and subsequent washing, the occurrence and evenamount of immunocomplex formation may be determined by subjecting sameto a second antibody having specificity for the first. Of course, inthat the test sample will typically be of human origin, the secondantibody will preferably be an antibody having specificity in generalfor human IgG. To provide a detecting means, the second antibody willpreferably have an associated enzyme that will generate a colordevelopment upon incubating with an appropriate chromogenic substrate.Thus, for example, one will desire to contact and incubate theantisera-bound surface with a urease or peroxidase-conjugated anti-humanIgG for a period of time and under conditions which favor thedevelopment of immunocomplex formation (e.g., incubation for 2 hours atroom temperature in a PBS-containing solution such as PBS-Tween).

After incubation with the second enzyme-tagged antibody, and subsequentto washing to remove unbound material, the amount of label is quantifiedby incubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid [ABTS]and H₂ O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generation, e.g.,using a visible spectra spectrophotometer.

Vaccine Preparation and Use

Immunogenic compositions, proposed to be suitable for use as a vaccine,may be prepared most readily directly from immunogenic OMP proteinsand/or peptides prepared in a manner disclosed herein. Preferably theantigenic material is extensively dialyzed to remove undesired smallmolecular weight molecules and/or lyophilized for more ready formulationinto a desired vehicle.

The preparation of vaccines which contain peptide sequences as activeingredients is generally well understood in the art, as exemplified byU.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792;and 4,578,770, all incorporated herein by reference. Typically, suchvaccines are prepared as injectables. Either as liquid solutions orsuspensions: solid forms suitable for solution in, or suspension in,liquid prior to injection may also be prepared. The preparation may alsobe emulsified. The active immunogenic ingredient is often mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the vaccine may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,or adjuvants which enhance the effectiveness of the vaccines.

The vaccines are conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkalene glycols or triglycerides: such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5% to 10%, preferably 1-2%. Oral formulations include such normallyemployed excipients as, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate and the like. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain 10-95% of active ingredient,preferably 25-70%.

The proteins may be formulated into the vaccine as neutral or saltforms. Pharmaceutically acceptable salts, include the acid additionsalts (formed with the free amino groups of the peptide) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups mayalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective andimmunogenic. The quantity to be administered depends on the subject tobe treated, including, e.g., the capacity of the individual's immunesystem to synthesize antibodies, and the degree of protection desired.Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner. However, suitable dosage ranges areof the order of several hundred micrograms active ingredient pervaccination. Suitable regimes for initial administration and boostershots are also variable, but are typified by an initial administrationfollowed by subsequent inoculations or other administrations.

The manner of application may be varied widely. Any of the conventionalmethods for administration of a vaccine are applicable. These arebelieved to include oral application on a solid physiologicallyacceptable base or in a physiologically acceptable dispersion,parenterally, by injection or the like. The dosage of the vaccine willdepend on the route of administration and will vary according to thesize of the host.

Various methods of achieving adjuvant effect for the vaccine includesuse of agents such as aluminum hydroxide or phosphate (alum), commonlyused as 0.05 to 0.1 percent solution in phosphate buffered saline,admixture with synthetic polymers of sugars (Carbopol) used as 0.25percent solution, aggregation of the protein in the vaccine by heattreatment with temperatures ranging between 70° to 101° C. for 30 secondto 2 minute periods respectively. Aggregation by reactivating withpepsin treated (Fab) antibodies to albumin, mixture with bacterial cellssuch as C. parvum or endotoxins or lipopolysaccharide components ofgram-negative bacteria, emulsion in physiologically acceptable oilvehicles such as mannide mono-oleate (Aracel A) or emulsion with 20percent solution of a perfluorocarbon (Fluosol-DA) used as a blocksubstitute may also be employed.

In many instances, it will be desirable to have multiple administrationsof the vaccine, usually not exceeding six vaccinations, more usually notexceeding four vaccinations and preferably one or more, usually at leastabout three vaccinations. The vaccinations will normally be at from twoto twelve week intervals, more usually from three to five weekintervals. Periodic boosters at intervals of 1-5 years, usually threeyears, will be desirable to maintain protective levels of theantibodies. The course of the immunization may be followed by assays forantibodies for the supernatant antigens. The assays may be performed bylabeling with conventional labels, such as radionuclides, enzymes,fluorescers, and the like. These techniques are well known and may befound in a wide variety of patents, such as U.S. Pat. Nos. 3,791,932;4,174,384 and 3,949,064, as illustrative of these types of assays.

EXAMPLE I EDTA-Based Extraction of Outer Membrane Fragments

In order to obtain antibody to the OMP antigens, outer membranefragments from M. catarrhalis strain 035E were prepared as an immunogen.M. catarrhalis strain 035E cells were grown on agar plates using brainheart infusion broth. Plates were incubated at 37° C. in a candleextinction jar. Outer membrane fragments were subsequently prepared fromthese cells by the EDTA-based extraction procedure of Murphy et al.,Microb. Path., 1989.

EXAMPLE II Isolation of M. catarrhalis OMPs

In light of the present disclosure's identification of monoclonalantibodies specific to selected M. catarrhalis OMPs, it is proposed thatthe corresponding OMP antigen may be purified using the followinggeneral procedure. Cell envelopes will be prepared by sonication orouter membrane fragments will be extracted by EDTA-based treatment ofwhole M. catarrhalis cells. These membranes will be treated with ionicor non-ionic detergents to release the desired proteins which can thenbe purified by using conventional column chromatography or byimmunoaffinity techniques.

EXAMPLE III Preparation of Monoclonal Antibodies Specific for M.catarrhalis Outer Membrane Proteins

The present example illustrates the steps employed by the inventors inreducing certain aspects of the invention to practice. In particular,this example relates to the generation and identification of hybridomasthat produce monoclonal antibodies to the 30, 80 or 100 kD OMP antigen.Once hybridomas secreting monoclonal antibodies to surface-exposed OMPantigens from M. catarrhalis were identified, those determined toproduce antibody to these OMP antigens were selected and cultured toproduce antibody for use in other studies, such as those involvingpulmonary clearance of M. catarrhalis.

BALB/c mice were immunized by intraperitoneal injection with outermembrane fragments of M. catarrhalis strain 035E prepared by theEDTA-based extraction procedure. Each animal was immunized with 50-100μg protein in 0.1 ml of Freund's complete adjuvant. One month later, theanimals were boosted with an identical quantity of this same proteinpreparation in incomplete Freund's adjuvant. Three weeks later, the micewere given an intravenous injection (into the tail vein) with 50 μgprotein of the same membrane preparation suspended in PBS.

The "pancake" fusion method was employed as follows:

SP_(2/0) -Ag14 plasmacytoma cells were employed. These cells weremaintained in DMEM (Dulbecco's Modified EagleMedium)/Penicillin-Streptomycin-Glutamine with 15% fetal bovine serum,1% Fungizone and 8-azaguanine. Two weeks prior to the fusion, some ofthe cells were split into media with 1% Fungizone but lacking8-azaguanine. These cells were maintained for 10 days at a density of nogreater than 1-2×10⁵ /ml. Beginning three days before the fusion,SP_(2/0) cells were subcultured every 24 hours and maintained at anapproximate density of 2-3×10⁵ /ml. Three days before the fusion, themice were boosted intravenously with about 50 μg of protein immunogen.On the day of the fusion, two mice were sacrificed by cervicaldislocation. The spleens were removed aseptically and macerated. Spleencells were collected in 10 mls of DMEM-HY media (60 ml NCTC-109, 6 tubeshypoxanthine-thymidine-glycine stock soln., 6 tubes oxalaceticacid-bovine insulin stock soln., 12 mlpenicillin-streptomycin-glutamine, 2.7 ml 100 mM Na pyruvate, and 508 mlDMEM). At room temperature, SP_(2/0) cells and spleen cells werecollected by centrifugation at 170×g for 11 min. in their respectivetubes. SP_(2/0) cells and spleen cells were each resuspended in a totalof 5 mls of DMEM-HY media.

The hypoxanthine-thymidine-glycine stock solution was prepared by adding136 mg hypoxanthine to 100 ml 0.1M HCl, 38.7 mg thymidine to 100 ml H₂O, and 2.3 mg glycine to 20 ml H₂ O. These solutions were dissolvedseparately, combined and then aliquoted into 2.2 ml volumes,

The oxalacetic acid-bovine insulin stock solution was prepared bydissolving 80.3 mg bovine insulin in 100 ml H₂ O, adding 1.32 gmoxaloacetic acid and aliquoting into 1 ml. volumes.

Spleen cells were then diluted to 2×10⁸ cells/5 mls and the SP_(2/0)cells was diluted to 2×10⁷ cells/5 mls. The ratio of spleen cells toSP_(2/0) cells was 10:1. Spleen cells were then mixed with SP_(2/0)cells in a ratio of 1:1. The spleen-SP_(2/0) mixture was then treatedwith 3 mls of 50% PEG/DMEM-HY media for 35 sec. Fused spleen-SP_(2/0)cells were washed immediately with DMEM-HY and incubated in 30% HY:HIFCS(35 ml DMEM-HY, 15 ml FBS, filter) for 24 hours at 37° C. 24 hours afterthe fusion, media and fused cells were collected in 20% HY:HIFCS (80 mlDMEM-HY, 20 ml FBS, filter) by centrifugation at 170×g for 5 min. Thefused cells were then resuspended in 100 mls of 20% HAT:HIFCS andtransferred to 96-well microtiter plates, 100 μl/well. One week afterthe fusion, 100 μl of 20% HY:HIFCS were added to each well. Two weeksafter the fusion, when wells containing proliferating hybrid cellsbecame acidic, each positive well was split into a 2 ml well on a24-well plate and the culture supernatant assayed for antibodycharacterization.

Supernatants from these clones were screened for antibodies to M.catarrhalis by ELISA binding and Western blot methods usingEDTA-extracted outer membrane fragments of M. catarrhalis strain 035E asantigen for the ELISA, and whole cell lysates of this strain as antigenfor Western blots. Positive supernatants were then tested by theindirect antibody accessibility RIA to investigate the surface exposureof outer membrane antigens as described by Kimura et al. (1985 and1986).

Positive hybridomas were then cultured in standard DME and themonoclonal antibodies were purified from culture supernatants on ProteinA--Sepharose CL-4B as described by Ey et al., 1978.

Each Mab identified as being reactive with M. catarrhalis in Westernblot analysis was used in the indirect antibody accessibility assay todetermine if these Mabs were reactive with surface-exposed determinantsof this organism. The antibody accessibility assay performed wasdescribed by Patrick et al., 1987.

Mab 10F3, which reacted with an antigen with an apparent MW ofapproximately 80,000 in Western blot analysis, was shown to bind to thesurface of whole cells of strain 035E. This Mab reacted with 4 of 10different M. catarrhalis strains tested in colony blot-RIA analysis bythe method of Gulig et al., 1987. A culture deposit of hybridomassecreting Mab 10F3 has been made with the American Type CultureCollection as ATCC accession number HB 11092.

Mab 17C7 reacted with two different size bands in Western blot analysis.This Mab reacted with a band near the top of the gel that migrated in adiffuse form and sometimes with a second band that migrated with anapparent MW of 100,000. For the purpose of clarity, the Mab will bedefined as being reactive with the 100,000 kD antigen. This Mab bound tothe surface of strain 035E and reacted with all ten different M.catarrhalis strains tested in the colony blot RIA. A culture deposit ofhybridomas secreting Mab 17C7 has been made with the American TypeCulture Collection as ATCC accession number HB 11093.

Mab 8B6 reacted with an antigen with an apparent MW of approximately30,000 in Western blot analysis. This Mab was also reactive with thesurface of strain 035E and reacted with all ten different M. catarrhalisstrains tested in the colony blot-RIA. A culture deposit of hybridomasMab 8B6 has been made with the American Type Culture Collection as ATCCaccession number HB 11091.

EXAMPLE IV Pulmonary Clearance of M. catarrhalis using MonoclonalAntibodies Specific for the 30, 80 and 100 kD OMPs

The present example illustrates steps employed by the inventors inreducing certain aspects of the invention to practice. This exampledemonstrates the ability of monoclonal antibodies to the 30, 80 and 100kD OMPs to enhance pulmonary clearance of M. catarrhalis using a murinemodel system. Thus, this example demonstrates that antibodies to the 30,80 or 100 kD OMP may be useful for passive immunization and thatvaccines comprising these OMPs are likely to provide active immunityagainst M. catarrhalis infections.

A. Antibody Administration

Eighteen hours prior to bacterial challenge, groups of 5 mice werepassively immunized by intravenous administration of monoclonal antibody17C7, 8B6 or 10F3. Control animals were immunized with an irrelevantantibody, 2H11, which was directed against an outer membrane protein ofHaemophilus ducreyi. Each animal received an equivalent amount ofpurified antibody corresponding to 150 μg of total protein.

B. Method of Bacterial Inoculation

Mice were anaesthetized by intramuscular injection of 2 mg of ketamineHCL (Fort Dodge Lab, Fort Dodge, Iowa) and 0.2 mg of acepromazinemaleate (Fort Dodge Lab). After tracheal exposure each animal wasintubated transorally with a 20 gauge intravenous catheter which wasadvanced until it could be visualized through the translucent trachealwall. A PE-10 polyethylene tube containing 5 μl of bacterial suspensionwas then passed through the catheter into the lung where the bacteriawere deposited with 150 μl of air. This technique delivered the inoculumto a localized, peripheral segment of the lung. In all experiments, micewere challenged with M. catarrhalis strain 035E.

C. Pulmonary Clearance

In each experiment, 5 mice were sacrificed by intraperitoneal injectionof 0.75 mg of sodium pentobarbital (Abbott Labs, Chicago, Ill.)immediately after inoculation (0 h), to determine bacterial depositionin the lungs. At 6 hours after challenge, experimental (17C7-, 8B6- or10F3-immunized) and control (2H11 immunized) groups were sacrificed, andthe number of viable bacteria remaining in the lungs was determined asfollows: the lungs from each animal were removed aseptically andhomogenized in 2 ml of sterile BHI broth in a tissue homogenizerfollowed by grinding in a tissue grinder until smooth. The homogenatewas serially diluted in BHI broth, plated on BHI agar and incubated at37° C. in an air incubator with a 5% CO₂ atmosphere for 24 h. Clearanceof M. catarrhalis from the lungs is expressed as the percentage ofcolony forming units (cfu) remaining in the lung at each time pointcompared with the mean cfu of bacteria present at 0 h in the sameexperiment.

RESULTS

The mean number of viable bacteria remaining in the lungs of immunizedand control mice after bolus deposition of 0.98×10⁵ to 2.0×10⁵ cfu of M.catarrhalis 035E was determined and expressed as a percentage of theinitial inoculum.

                  TABLE I                                                         ______________________________________                                                        % of Bacteria Remaining                                       Immunization    at 6 h Post-Challenge                                         Regimen         Expt. #1   #2                                                 ______________________________________                                        No immunization 134        109                                                2H11 immunization                                                                             113        108                                                17C7 immunization                                                                             27         22                                                 8B6 immunization                                                                              32         45                                                 10F3 immunization                                                                             10         13                                                 ______________________________________                                    

EXAMPLE V Cloning the Gene Encoding the 80 kD OMP (10F3-Reactive) fromM. catarrhalis

The present Example illustrates steps employed by the inventor incloning the gene encoding for the 80 kD OMP from M. catarrhalis. Thepresent Example discloses one or more preferred recombinant E. coliclones, expressing the 80 kD OMP antigen, isolated by the followingprocedures.

A. Isolation of genomic DNA

M. catarrhalis strain 035E was used as a representative Moraxellapathogen in this study. Genomic DNA from M. catarrhalis strain 035E wasextracted and purified as follows. M. catarrhalis cells (approximately 2gms wet weight) were scraped from agar plates and resuspended in 20 mls.PBS. To this suspension was added 3.2 ml 10% (w/v) SDS and 1 ml RNase(10 mg/ml). This mixture was incubated at 37° C., then 3 mg proteinase Kadded, followed by further incubation at 55° C. overnight. The incubatedmixture was then extracted once with phenol, twice withphenol:chloroform:isoamyl alcohol, and three times withchloroform:isoamyl alcohol. The resulting DNA was then precipitated withtwo volumes of absolute ethanol, and collected with a Pasteur pipet.

B. Preparation of an M. catarrhalis genomic library in pBR322

The partial digestion of genomic DNA was achieved by incubating 100 μgportions of M. catarrhalis genomic DNA with varying amounts of therestriction enzyme PstI in a reaction volume of about 1.5 ml. at 37° C.for 1 hr. The partially digested genomic DNA was then size fractionatedby sucrose density gradient centrifugation. Fractions containing DNAfragments from about 6 kb to 23 kb in length were selected and dialysedto obtain purified genomic DNA fragments for ligation with pBR322.

The plasmid vector pBR322 was fully digested with PstI by incubating 15μg portions of pBR322 with 50 units of PstI in a 100 μl reaction volumeat 37° C. for 18 hrs. Ligation of the purified DNA fragments into thePstI-digested pBR322 vector was accomplished by incubating 300 ng of thepurified DNA fragments and PstI-digested pBR322 together with ATP and T4DNA ligase under conditions described by Maniatis et al. (1982). Afterligation, the DNA was diluted 1:5 with 10 mM TRis-HCl (pH 8.0) and wasused to transform E. coli RR1 made competent by the CaCl₂ method.

C. Screening transformed RR1 colonies by colony blot-radioimmunoassayfor M. catarrhalis OMP expression

The colony blot RIA was accomplished as described by Gulig et al. (1987)with monoclonal antibody 10F3 as the primary antibody.

D. Characterising recombinant E. coli clones expressing M. catarrhalisOMP antigens

Clones which reacted with monoclonal antibody 10F3 in the colony blotRIA were cultured using LB medium containing the antibiotic tetracycline(15 μg/ml). Whole cell lysates of recombinant E. coli RR1 expressing M.catarrhalis OMP antigens were prepared as described by Patrick et al.,1987. Briefly, portions of these whole-cell lysates were subjected toSDS-PAGE as described in Gulig et al., 1987, and then stained withCoomassie blue or transferred to nitrocellulose for Western blotanalysis. The results are shown in FIG. 1 which indicate thatrecombinant 80 kD OMP gene is expressed in the clones identified bymonoclonal antibody 10F3.

EXAMPLE VI Cloning of the Genes Encoding the 30 kD (8B6-Reactive) and100kD (17C7-Reactive) Outer Membrane Proteins of M. catarrhalis

A. Isolation of genomic DNA

M. catarrhalis genomic DNA was isolated from strain 035E as describedabove.

B. Preparation of a M. catarrhalis genomic DNA library using thebacteriophage vector λGEM-11

Purified M. catarrhalis DNA (100 μg) was partially digested with Sau3A(Promega Biotech) at room temperature as described above. The digestedDNA was size-fractionated in sucrose density gradients and fragments ofDNA 15 kb and larger were collected for use in library construction.These DNA fragments (1 μg) were filled in using the Klenow procedure(Promega) at 14° C. for 90 min. These fragments were then cleaned bystandard procedures and ligated onto the phage DNA arms and packagedusing the protocol and reagents supplied by Promega in theLambdaGEM--11* Xho I Half-Site Arms Cloning System, except that T4 DNAligase from BRL was used. After packaging, the phage-based library wastitered using E. coli LE392. This genomic library contained 50,000recombinant clones.

C. Screening of the bacteriophage-based genomic DNA library withmonoclonal antibodies 17C7 and 8B6

20,000 plaques were screened with Mabs 17C7 and 8B6 using the plaquescreening procedure described in Current Protocols in Molecular Biology(Wiley Interscience) using radioiodinated goat anti-mouse Ig as theprobe to detect Mabs bound to plaque material. One recombinant phagereactive with each Mab was ultimately identified.

D. Characterization of the recombinant phages reactive with Mabs 17C7and 8B6

Liquid lysate cultures of these recombinant phage were prepared by thestandard methods described in Current Protocols in Molecular Biology.The DNA was extracted using standard methods.

The recombinant phage reactive with Mab 17C7 had a DNA insertapproximately 11 kb in size. The recombinant phage reactive with Mab 8B6had a DNA insert approximately 18 kb in size.

Phage harvested from liquid lysates were heated at 100° C. for 3 min. instandard SDS digestion buffer and then used for SDS-PAGE and Westernblot analysis to confirm that these recombinant phage were expressingthe appropriate M. catarrhalis antigens.

FIG. 2 is an illustrative Western blot analysis of proteins from E. coliclone LE392/8B6, which expresses the 30 kD OMP antigen. In this study,the various indicated samples were subjected to PAGE, transferred to anitrocellulose membrane, and probed with the 30 kD OMP-specificmonoclonal antibody 8B6. As can be seen, a band having an approximatemolecular weight of 30 kD is seen in the LE 392/8B6 lane (lane C), and asimilar band is seen in the positive control lane (lane F). The natureof the two additional bands seen in the LE 392/8B6 lane (lane C) isunclear, but they could be due to processing of the recombinant proteinor overloading of the gel. The bands seen in the negative control lanes(lanes D and E) are clearly due to spillover from lanes C and F.

FIG. 3 shows a similar Western blot analysis of a phage lysate from aclone expressing the 100 kD OMP, designated LE 392/17C7, probed withmonoclonal antibody 17C7. Lanes C-E comprise phage lysate proteins fromclone LE392/17C7. These lanes exhibit slight reactivity in the 100 kDrange, that comigrate with an apparently corresponding 100 kD band inthe positive control (lane H). It is noted, however, that the majorityof reactivity in the lysate samples migrated at an apparently highermolecular weight, possibly due to protein aggregation, lack ofprocessing or a similar phenomenon.

The present invention has been described in terms of particularembodiments found or proposed by the present inventors to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. For example, due to codon redundancy, changescan be made in the underlying DNA sequence without affecting the proteinsequence. Moreover, due to biological functional equivalencyconsiderations, changes can be made in protein structure withoutaffecting in kind or amount of the biological action. All suchmodifications are intended to be included within the scope of theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. An antigen composition comprising an Moraxellacatarrhalis outer membrane antigen selected from the group consisting ofM. catarrhalis outer membrane antigens immunologically reactive withmonoclonal antibody 10F3 (ATCC HB 11092) or 17C7 (ATCC 11093), whereinsaid antigen is purified free of other M. catarrhalis outer membraneantigens.
 2. The composition of claim 1, wherein the selected M.catarrhalis antigen is immunologically reactive with monoclonal antibody10F3 (ATCC HB 11092).
 3. The composition of claim 1, wherein theselected M. catarrhalis antigen is immunologically reactive withmonoclonal antibody 17C7 (ATCC HB 11093).
 4. The composition of claim 1,wherein the M. catarrhalis antigen is prepared by recombinant means. 5.The composition of claim 1, further defined as being essentially free ofM. catarrhalis antigens other than those reactive with monoclonalantibody 10F3 or 17C7.
 6. The composition of claim 1, wherein theantigen is comprised in a pharmaceutically acceptable buffer.
 7. Thecomposition of claim 6, wherein the pharmaceutically acceptable bufferincludes an acceptable carrier or adjuvant.